[RASMB] Re: XL-A/I temp control

Fri Dec 17 10:16:00 PST 2004

In my 2 cent worth of opinion, this what I believe:

 If a protein or polymer understudy can not withstand a degree
temperature variation in the instrument while data is collecting because
of the stability issue, then I think AUC is not a technique of choice
for undertake the study. I am no expert in AUC but after few years of
running this instrument and doing the analysis and spent a lot of my
spare time to check the reproducibility between same sample and between
instruments (we have two) and analyzed and compare the collected raw
data using different softwares; I come to the conclusion that the
biggest culprits in analyzed the result is the V-bar and density issue.
Therefore I think the energy and resource could be better spent on
compile a more complete list of commonly used compounds that are used
for buffer solution. I wish that for those of you who has the means and
would like to do some useful service to our community, can create a
project, to get some undergraduate student who is interested in AUC
technique, to compile these useful information so David Hay, John philo
and Tom Laue can update and complete (maybe never) the SEDTERP buffer
data list(the next best thing for average user). I agree wholeheartedly
with John Correia, Beckman should joined up with Anton Paar to bundle
Density Meters into XLA/XLI quotes. This way the analyze result will be
and should be very much the same from instrument to instrument and from
lab to lab. 
20 years ago when protein sequencing was hot and DNA sequence was not.
Applied Biosystem, did the similar thing by sending an unknown sample to
the interested lab around the country to see which lab is getting the
unknown sequence correctly.  I remember there are about 20 labs
participating in the fun and only 10%-20% got the sequence right.
Borries Demeler's suggestion is not new.  Back in 1999 and 2001, Preston
Hensley has put out a call for similar purpose (28 Jun 1999, an AUC test
system, 25 Aug 2001, Methods calibration study - a call).  I do not know
what came out of the call. Even though AUC is a beautiful technique that
does not need a comparison with any standards.  I find it is useful to
have a standard protein with a simple monomer-dimer system around so
whenever I have doubt about the result, I can always go back to run my
standard protein to see if anything wacky happens(if it is not yet
obvious!). Provided I use the same analysis software, same v-bar,
temperature and in addition the same edited radius and same speed.  For
SV, using the same group # of scans and same density and viscosity
value. I constantly remind people who use our facility and who is not
familiar with this technique that AUC is not a masspectrometry, if one
need accurate mw information, one need to go to masspectrometry facility
to get that information. 
In short, looking at the bigger picture, when one employs this technique
all one wants to ask is do you have an association system?  If it is on
the borderline then one need to find another technique to support one's
conclusion. As I mention before if a protein understudy can not
withstand a degree in temperature variation because of the stability
issue, then I think AUC is not a technique of choice for undertaken the
study.  This is only my opinion, a protein sequencer turn AUC user.
When I first started to use AUC, I had done some test to see how
important of Vbar and Rho could affect the outcome of a SE analysis.  I
found out that a 10% error in the value of vbar or rho could result in
about 40% error (using Beckman origin software) in the apparent
molecular mass obtained from the SE curve.  Even a 3% error in the
measurement of vbar or rho could result in drawing the misleading
conclusion that association had occurred in the macromolecular where it
had not or vice versa.

Kind regards

Chris

-------------------------------------------------------------

Christopher Chin

Manager

Sealy Center for Structural Biology

HBC&G, 5134 MRB. rt1055

UTMB cchin at utmb.edu, 

409-772-1693, efax 630-604-3416

-------------------------------------------------

-----Original Message-----
From: rasmb-admin at server1.bbri.org [mailto:rasmb-admin at server1.bbri.org]
On Behalf Of JA KORNBLATT
Sent: Friday, December 10, 2004 9:22 AM
To: Arthur Rowe
Cc: mchien at beckman.com; rasmb at server1.bbri.org
Subject: Re: [RASMB] Re: XL-A/I temp control

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jack kornblatt would be in favour of arthur's and borries' suggestion.
jack

> "As an approach to the size of the problem, would there be support for

> Borries Demeler's suggestion (a single sample to be circulated and 
> multiple users on multiple machines to report an s value under defined

> conditions)? After all, the NCMH + Borries's Lab gives us 6 machines 
> for starters."
>

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